The use of carbohydrate and peptide antigens in vaccines is greatly hampered by their lack of immunogenicity when injected directly into a patient. Such antigens, when injected alone, are usually ignored by antigen-presenting cells (APCs), cleared rapidly and do not induce an immune response.
In most cases, it is also necessary to administer the antigen in combination with an adjuvant. The adjuvant may be a simple delivery system such as liposomes, which slow clearance of the antigen and make it more likely to reach and be taken up by APCs. However, this in itself is not very effective and usually needs to be combined with agents that stimulate the immune system, such as bacterial products which stimulate cytokine formation. Cytokines themselves may also be co-administered. Many of these products are too toxic or too experimental to be used in humans, and the most effective adjuvants are not approved for human use. Most of the adjuvants available for use in humans are of limited effectiveness. Finding effective adjuvants suitable for human use is a continuing challenge.
Carbohydrate antigens are of particularly weak immunogenicity because they can stimulate only B-cell and not T-cell responses. This is usually addressed by conjugating the carbohydrate to a protein carrier. However, in order to raise an immune response it is also necessary to use an adjuvant.
Carbohydrate antigens are potential targets for anti-cancer immunotherapy since they are exposed at the surface of tumour cells but hidden on normal cells. Many bacteria and other pathogens are also distinguished by carbohydrate antigens which would be a good target for vaccines, if carbohydrates were not so poorly immunogenic. Improving the immunogenicity of carbohydrate antigens would thus have applications in a wide variety of therapeutic fields.
Cancer cells almost always are glycosylated in an aberrant manner, a characteristic that distinguishes them from normal cells. (Glycoconjugate J. (1997), 14:569; Adv. Cancer Res. (1989), 52:257; Cancer Res. (1996), 56:5309). In most cases, the aberrant glycosylation is presented on the surface of the cells in the form of glycoproteins and glycolipids. These altered carbohydrate structures can therefore be called tumour associated antigens (TAA), which often do not occur on normal cells. In many cases, cells do not show a homogeneous glycosylation, i.e. different glycoforms of complex glycan-chains exist on one cell surface (Annu. Rev. Biochem. (1988), 57:785).
In the course of the discovery and the subsequent characterisation of the most varied tumour associated antigens, research has shown that they have important functions for the cancer cells. For example, the tumour associated antigens enable the degenerate cells to show properties characteristic of the malignant phenotype, such as an increased capability for adhesion, which play an important role in establishing metastases. However, such antigens can at certain stages also be expressed on normal cells where they are responsible for the normal functions of these cells. Thus, tumour associated antigens are structures which are predominantly presented by tumour cells, generally on or in the cell membrane, and which allow them to be differentiated from non-malignant tissue. Tumour associated antigens may be, for example, polypeptides, in particular glycosylated proteins, or glycosylation patterns of polypeptides. Other structures which may represent a tumour associated antigen include glycolipids, for example, gangliosides, such as GM2. Such tumour associated antigens may be represented by changes in the composition of lipids of the cell membrane which may be characteristic of cancer cells. Tumour associated antigens include the following examples.
N-CAM (Neuronal Cell Adhesion Molecule), which is often expressed on tumours of neuronal origin and which effects homophilic adhesion (J. Cell Biol. 118 (1992), 937).
The Lewis Y carbohydrate antigen, which occurs on the majority of tumours of epithelial origin, but which also plays an important role during the fetal development of epithelial tissues. It has been shown that the expression of this antigen in lung cancer is strongly associated with an unfavourable prognosis since Lewis Y positive cancer cells obviously have a higher metastatic potential (N. Engl. J. Med. 327 (1992), 14).
CEA (Carcino Embryonic Antigen), which often occurs on epithelial tumours of the gastrointestinal tract and which has been identified as a self-adhesion molecule (Cell 57 (1989), 327).
Ep-CAM (Epithelial Cell Adhesion Molecule), which is expressed on nearly all tumours of epithelial origin, but which also occurs on a large number of normal epithelia. It has been characterized as a self-adhesion molecule and can therefore be classified as a pan-epithelial adhesion antigen (J. Cell Biol. 125 (1994), 437).
Further examples of tumour-associated antigens are Sialyl Tn carbohydrate, Lewis antigens (Lewis-x, Lewis-b, Lewis y-structures), Globo H carbohydrate, gangliosides such as GD2/GD3/GM2, Prostate Specific Antigen (PSA), CA 125, CA 19-9, CA 15-3, TAG-72, EGF receptor, Her2/Neu receptor, p97, CD20 and CD21. Monoclonal antibodies directed against all these antigens are available. Examples of tumour associated antigens are described in DeVita et al. (Eds., “Biological Therapy of Cancer”, 2. Edition, Chapter 3: Biology of Tumor Antigens, Lippincott Company, ISBN 0-397-51416-6 (1995), (Elektrophoresis (1999), 20:362; Curr. Pharmaceutical Design (2000), 6:485, Neoplasma (1996), 43:285)).
There are various methods of treatment of cancer, yet the success rate of the present treatments regimens is still to be improved. Aside from surgery and chemotherapy, immunotherapeutic treatment is also known.
In passive immunotherapy, monoclonal antibodies (MAbs) are administered systemically to a patient in a suitable amount to directly bind to a target. The aim of the treatment is to form an immune complex and through a series of immune reactions the cell or organism afflicted with the target is killed. The therapeutic effect is depending on the concentration of the MAbs in the circulation and their biological half-life, which is usually quite short. It is therefore necessary to repeat the administration within an appropriate timeframe. If xenogeneic MAbs, such as murine antibodies, are used adverse reactions are expected, possibly leading to anaphylactic shock. Because of this drawback, such immunotherapies are employed for a limited time only.
Active immunization regimens activate the immune system of patients in a different way. Following the administration of an antigen that resembles a specific target, the patient's humoral and T-cell specific immune response induces defence mechanisms to combat the target in vivo. Vaccine antigens of various types and against a wide variety of different diseases are well known in the art. For example, vaccination against Hepatitis B using vaccines containing surface hepatitis B antigens are well known. It has been shown that high dose ranges of antigens used for vaccination of as well as low dose vaccination can give sufficient rates of seroconversion (Parish D. C. et al., 1991, Southern Medical Journal, 84, 426-430; Goudeau A. et al., 1984, The Lancet, 10, 1091-1092).
Mannan-mucin fusion proteins are also known and can be used for generating cytotoxic T cells. It has been shown that depending on the dosage of the administered fusion protein to mice, either almost only cellular immunity (low doses) or only humoral immunity (high doses) can be induced (Pietersz G. A. et al., 1998, Cancer Immunol. Immunother., 45, 321-326).
For active immunization, the antigens are usually presented in an immunogenic formulation to provide a vaccine. Antigens mimicking the targets have either similarities in the primary and secondary sequence of the targets or fragments thereof. Mimotopes or mimotopic antigens, however, have similarities in the tertiary structure of the target.
Although many products have been developed for the treatment of cancer there is still a high demand on providing substances which have improved characteristics compared to the already known substances. In particular, in the field of vaccination, there is a need in products that are highly immunogenic, easily reproducible and highly effective, but do not cause severe side effects.
WO 02/32404 (Consejo Superior de Investigaciones Scientificas) discloses nanoparticles formed from metal or semiconductor atoms in which ligands comprising carbohydrates are covalently linked to the core of the nanoparticles. These nanoparticles are used for modulating carbohydrate mediated interactions and are soluble and non-toxic. PCT application claiming priority from GB-A-0313259.4 (Consejo Superior de Investigaciones Scientificas and Midatech Limited) discloses magnetic nanoparticles having cores comprising passive and magnetic metal atoms, the core being covalently linked to ligands. GB application 0411537.4 (Consejo Superior de Investigaciones Scientificas and Midatech Limited) discloses nanoparticles including magnetic nanoparticles which are conjugated to RNA ligands, in particular siRNA ligands.